Screening for inhibitors of M. tuberculosis persistence-related lipid metabolism

结核分枝杆菌持久性相关脂质代谢抑制剂的筛选

• 批准号: 8509336
• 负责人: Robert B Abramovitch
• 金额: $ 19.01万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-08 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8509336
• 关键词: Age; Air; American; Animal Model; Animals; Anti-Inflammatory Agents; Anti-inflammatory; Antibiotics; Bacteria; Biological Assay; Carbon Monoxide; Cessation of life; Chronic; Clinical; Communicable Diseases; Cues; DNA Binding; Development; Drug resistance; Environment; Exhibits; Fluorescence; Gene Expression; Genes; Goals; Granuloma; Growth; HIV; HIV Infections; Health; Human; Hypoxia; Immune; Immune response; Immune system; Immunity; In Vitro; Inclusion Bodies; Infection; Lead; Libraries; Lipid Inclusion; Lipids; Maintenance; Malnutrition; Measures; Metabolism; Mission; Modeling; Multi-Drug Resistance; Mycobacterium tuberculosis; National Institute of Allergy and Infectious Disease; Nitric Oxide; Nutrient; Oxygen; Pathway interactions; Phagosomes; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phase; Phosphotransferases; Physiology; Play; Population; Predisposition; Recycling; Regimen; Reporter; Role; Sampling; Sputum; Staging; Staining method; Stains; Structure-Activity Relationship; System; Triglycerides; Tuberculosis; aging population; cell killing; combat; disease transmission; global health; high throughput screening; inhibitor/antagonist; lipid metabolism; macrophage; nile red; pathogen; pre-clinical; pressure; public health relevance; resistant strain; response; screening; sensor; tuberculosis drugs

DESCRIPTION (provided by applicant): Infection with Mycobacterium tuberculosis (Mtb) often results in a chronic tuberculosis (TB) infection. Of those infected, ~90% mount a successful immune response that sequesters the bacterium in a granuloma. The granuloma limits the spread and growth of the bacterium and results in a persistent infection. Under conditions that weaken the immune system Mtb can be released from the granuloma and establish an active, infectious disease state. Developing new therapies that target mechanisms required for Mtb to establish, maintain or exit a persistent state has the potential to shorten the course of current ~6-9 month Mtb drug regimens as well as combat the emergence of multidrug-resistant Mtb. Mtb alters its physiology in response to host immune pressures thus enabling the bacterium to remain viable in humans for decades. Mtb is an intracellular pathogen that resides within macrophages (M), a host immune cell that kills most other bacteria. Following infection, the M releases immune modulators that orchestrate the formation of a granuloma around the infected M. The granuloma limits the availability of nutrients and oxygen to the bacterium and drives Mtb to realign its gene expression and physiology to support a non-replicative, persistent (NRP) state. Transition into and maintenance of NRP is governed, in part, by the DosRST two component regulator system (TCS). DosR is a DNA binding response regulator that is phosphorylated by the sensor kinase DosT to induce expression of approximately 50 genes in response to cues such as hypoxia. One of the most strongly DosR induced genes is triacylglycerol synthase 1 (tgs1), which is responsible for the accumulation of intracellular triacylglycerol (TAG). In vitro growth conditions that induce DosR and NRP cause Mtb to accumulate inclusion bodies that are primarily composed of TAG. These TAG inclusion bodies are also observed during infections and in clinical sputum samples from infected humans, supporting a role for TAG accumulation in the progression of TB during chronic infection. We propose to exploit Mtb TAG metabolism pathways to develop high throughput screening (HTS) platforms that target Mtb persistence pathways. We will develop fluorescent reporter assays that measure TAG metabolism: i) directly by staining TAG inclusions with the fluorescent stains, or ii) indirectly by using a synthetic tgs1'::GFP reporter strain (Specific Aim 1). These HTS platforms will be optimized and validated in a 384 well format and the best performing platform will be used for a HTS of a 500,000 small compound library for inhibitors of TAG metabolism (Specific Aim 2). Hits identified in the screen will be validated, prioritized and the most promising compounds will undergo preclinical characterization including, mechanism of action, structure activity relationship studies, and efficacy in animal models of infection (Specific aim 3). The goa of this proposal is to identify a lead compound that is suitable for further optimization and that specifically targets the chronic stage of tuberculosis infection.

描述(由申请人提供)：感染结核分枝杆菌(Mtb)通常会导致慢性结核病(TB)感染。在感染的人中，约90%的人成功地进行了免疫反应，将细菌隔离在肉芽肿中。肉芽肿限制了细菌的传播和生长，并导致持续感染。在削弱免疫系统的情况下，结核分枝杆菌可以从肉芽肿中释放出来，并建立一种活跃的传染病状态。开发针对结核分枝杆菌建立、维持或退出持续状态所需机制的新疗法，有可能缩短目前~6-9个月的结核分枝杆菌药物方案的疗程，并对抗多药耐药结核杆菌的出现。结核分枝杆菌会根据宿主的免疫压力改变其生理功能，从而使这种细菌能够在人类体内存活数十年。结核分枝杆菌是一种驻留在巨噬细胞(M)内的细胞内病原体，巨噬细胞是一种宿主免疫细胞，可以杀死大多数其他细菌。感染后，M释放免疫调节剂，在受感染的M周围形成肉芽肿。肉芽肿限制了细菌对营养物质和氧气的供应，并驱动Mtb重新调整其基因表达和生理，以支持非复制、持续(NRP)状态。向NRP的过渡和维护在一定程度上由DosRST双分量调节系统(TCS)管理。DosR是一种DNA结合反应调节子，被传感器激酶DOST磷酸化，以诱导大约50个基因的表达，以响应信号，如缺氧。DosR诱导作用最强的基因之一是三酰甘油合成酶1(TGS1)，它负责细胞内三酰甘油(Tag)的积累。在诱导DosR和NRP的体外生长条件下，Mtb积累了主要由Tag组成的包涵体。在感染期间和从感染者的临床痰样本中也观察到这些标签包涵体，支持标签积累在慢性感染期间结核病进展中的作用。我们建议利用结核分枝杆菌标签代谢途径来开发针对结核分枝杆菌持久性途径的高通量筛选(HTS)平台。我们将发展测量标签代谢的荧光报告试验：i)直接通过用荧光染料对标签包涵体进行染色，或ii)间接使用合成的TGS1：：GFP报告菌株(特定目标1)。这些HTS平台将在384井格式下进行优化和验证，性能最好的平台将用于用于标签代谢抑制剂(特定目标2)的500,000个小化合物库的HTS。筛选中确定的HITS将得到验证和优先排序，最有希望的化合物将进行临床前表征，包括作用机制、结构活性关系研究和感染动物模型的有效性(特定目标3)。这项提议的目标是确定一种适合进一步优化并专门针对结核病感染的慢性阶段的先导化合物。